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Title: Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

Abstract

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.

Authors:
 [1];  [2];  [3];  [4]
  1. Univ. of California, Santa Barbara, CA (United States). Bren School of Environmental Science and Management, Earth Research Inst., and Center for the Environmental Implications of Nanotechnology; National Institute of Chemical Physics and Biophysics, Tallinn (Estonia). Lab. of Environmental Toxicology
  2. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States). Biosystems and Biomaterials Division, Material Measurement Lab.
  3. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  4. Univ. of California, Santa Barbara, CA (United States). Bren School of Environmental Science and Management, Earth Research Inst., and Center for the Environmental Implications of Nanotechnology
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE: National Science Foundation (NSF); USEPA; Estonian Research Council
OSTI Identifier:
1377787
Report Number(s):
LLNL-JRNL-684658
Journal ID: ISSN 2079-4991; NANOKO; PII: nano6100181
Grant/Contract Number:  
AC52-07NA27344; PUTJD16
Resource Type:
Accepted Manuscript
Journal Name:
Nanomaterials
Additional Journal Information:
Journal Volume: 6; Journal Issue: 10; Journal ID: ISSN 2079-4991
Publisher:
MDPI
Country of Publication:
United States
Language:
English
Subject:
77 NANOSCIENCE AND NANOTECHNOLOGY; 54 ENVIRONMENTAL SCIENCES; 63 RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; Pseudomonas aeruginosa; Tetrahymena thermophila; carbon-14; sucrose; iodixanol; bioaccumulation; bioconcentration; Stokes' law

Citation Formats

Mortimer, Monika, Petersen, Elijah, Buchholz, Bruce, and Holden, Patricia. Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation. United States: N. p., 2016. Web. doi:10.3390/nano6100181.
Mortimer, Monika, Petersen, Elijah, Buchholz, Bruce, & Holden, Patricia. Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation. United States. doi:https://doi.org/10.3390/nano6100181
Mortimer, Monika, Petersen, Elijah, Buchholz, Bruce, and Holden, Patricia. Wed . "Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation". United States. doi:https://doi.org/10.3390/nano6100181. https://www.osti.gov/servlets/purl/1377787.
@article{osti_1377787,
title = {Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation},
author = {Mortimer, Monika and Petersen, Elijah and Buchholz, Bruce and Holden, Patricia},
abstractNote = {Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.},
doi = {10.3390/nano6100181},
journal = {Nanomaterials},
number = 10,
volume = 6,
place = {United States},
year = {2016},
month = {10}
}

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